Regulating stem cells

Abstract

Provided are methods for producing progenitor/precursor cells from a population of initiating cells (ICP) that have a density of less than 1.072 g/ml and at least 25% of which are CD31Bright by in vitro stimulating the ICP with different factors.

Claims

1. A method of making a composition, the method comprising: obtaining an initiating cell population (ICP) of at least 5 million cells that have a density of less than 1.072 g/ml, at least 1% of which are CD34+CD45/Dim, and at least 25% of which are CD31.sup.Bright; and in vitro stimulating the ICP by culturing the ICP in the presence of one or more factors selected from the group consisting of: autologous serum, bFGF, and IFN beta to differentiate into a progenitor/precursor cell population (PCP) comprising a subpopulation of cells that express one or more markers selected from the group consisting of: connexin 43, alfa actin, IgG1, IgG2a, and troponin.

2. The method according to claim 1, comprising preparing the PCP as a product for administration to a patient.

3. The method according to claim 1, comprising freezing the ICP prior to stimulating the ICP.

4. The method according to claim 1, comprising freezing the PCP.

5. The method according to claim 1, comprising transporting the PCP to a site at least 10 km from a site where the PCP is first created.

6. The method according to claim 1, wherein stimulating the ICP comprises: culturing the ICP in a first container during a first portion of a culturing period; removing cells of the ICP from the first container at the end of the first portion of the period; and culturing, in a second container during a second portion of the period, the cells removed from the first container.

7. The method according to claim 1, comprising locally administering the PCP to a site of the patient including injured tissue.

8. The method according to claim 7, wherein locally administering the PCP comprises implanting at the site a device including the PCP.

9. The method according to claim 8, comprising using the device to enable increased survival of PCP in injured tissue.

10. The method according to claim 8, comprising configuring the device for slow release of cells of the PCP into the injured tissue.

11. The method according to claim 8, comprising secreting, from the PCP, therapeutic molecules to the tissue.

12. The method according to claim 8, comprising secreting, from the device, soluble molecules that support the PCP.

13. A method of making a composition, the method comprising obtaining an initiating cell population (ICP) of at least 5 million cells that have a density of less than 1.072 g/ml, at least 1% of which are CD34+CD45/Dim, and at least 25% of which are CD31.sup.Bright; and in vitro stimulating the ICP by culturing the ICP in the presence of one or more factors selected from the group consisting of: autologous serum, bFGF, IFN beta, erythropoietin, a statin, an antidiabetic agent, a thiazolidinedione, rosiglitazone, a proliferation-differentiation-enhancing agent, anti-CD34, anti-Tie-2, anti-CD133, anti-CD117, LIF, EPO, IGF, M-CSF, GM-CSF, TGF alpha, TGF beta, VEGF, BHA, BDNF, GDNF, NGF, NT3, NT4/5, S-100, CNTF, EGF, NGF3, CFN, ADMIF, estrogen, prolactin, an adrenocorticoid hormone, ACTH, MCT-165, glatiramer acetate, a glatiramer acetate-like molecule, IFN alpha, glutamate, serotonin, acetylcholine, NO, retinoic acid (RA), insulin, forskolin, and cortisone to differentiate into a progenitor/precursor cell population (PCP) comprising a subpopulation of cells that express one or more markers selected from the group consisting of connexin 43, alfa actin, IgG1, IgG2a and troponin.

14. A method of making a composition, the method comprising obtaining an initiating cell population (ICP) of at least 5 million cells that have a density of less than 1.072 g/ml, at least 1% of which are CD34+CD45/Dim, and at least 25% of which are CD31.sup.Bright; and in vitro stimulating the ICP by culturing the ICP in the presence of one or more factors selected from the group consisting of: autologous serum, bFGF, IFN beta, anti-Tie-2, anti-CD133, and anti-CD117, VEGF, anti-VEGF, anti-VEGF receptor, heparin, MCDB-201, sodium selenite, dexamethasone, and BSA.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing results obtained from CCP cells in one representative experiment, in accordance with an embodiment of the present invention;

(2) FIG. 2 is a photograph showing morphology of angiogenic cell precursor cells, produced in accordance with an embodiment of the present invention;

(3) FIG. 3 is a photograph characterizing uptake of Ulex-Lectin and staining with CD31 stain of an ACP-rich PCP, produced in accordance with an embodiment of the present invention;

(4) FIG. 4 is a photograph characterizing uptake of Ac-LDL and staining with CD31 stain of an ACP-rich PCP, produced in accordance with an embodiment of the present invention;

(5) FIG. 5 is a photograph showing tube formation in an ACP-rich PCP, produced in accordance with an embodiment of the present invention;

(6) FIGS. 6A and 6B are graphs showing migration of Ac-LDL-DiO pre-labeled ACPs in response to hIL-8, in accordance with an embodiment of the present invention;

(7) FIG. 7 is a graph showing migration of Ac-LDL-DiO pre-labeled ACPs in response to a cultured medium, in accordance with an embodiment of the present invention;

(8) FIG. 8 is a graph of migration of PBMC cells in response to hIL-8, in accordance with an embodiment of the present invention;

(9) FIGS. 9A and 9B are graphs showing experimental results of improved ejection fraction and reduced necrosis in response to injection of ACP cells in accordance with an embodiment of the present invention;

(10) FIGS. 9C, 9D, and 9E are photographs showing sections taken from a rat's heart after injection of ACPs derived from a human-PBMC-derived CCP, produced in accordance with an embodiment of the present invention;

(11) FIG. 10 is a photograph showing the morphology of cardiomyocytes derived from the CCP and produced in accordance with an embodiment of the present invention;

(12) FIGS. 11A, 11B, and 11C are photographs showing immunostaining of CCP-derived cardiomyocytes, in accordance with an embodiment of the present invention;

(13) FIGS. 12A and 12B are graphs showing flow cytometry analysis results, obtained from immunostaining of a cardiomyocyte-rich PCP, in accordance with an embodiment of the present invention; and

(14) FIG. 13 is a graph showing experimental results of improved ejection fraction in a rat model of acute myocardial infarction following injection of the CCP-derived cardiomyocytes, in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Example 1

(15) A test was carried out in accordance with an embodiment of the present invention, and results are shown in Table 1 below. Peripheral blood was extracted from ten human volunteers for use in ten respective experiments. In each experiment, cells were fractioned from the blood using a Ficoll gradient in order to generate a population of peripheral blood mononuclear (PBMC) cells as source cells (S. cells). Subsequently, a CCP was generated in accordance with protocols described herein for Percoll based enrichment. Results in Table 1 show enrichment of the percentages of CD34+CD45/Dim cells in the CCP compared to the source cells. Enrichment is defined as the percentage of cells having a given characteristic in the CCP, divided by the percentage of cells having that characteristic in the source cells.

(16) TABLE-US-00001 TABLE 1 % CD34+ CD45/Dim Exp % Viability % CD45 Enrichment No S. cells CCP S. cells CCP S. cells CCP factor 1 97.56 97.86 94.00 93.46 1.4 4.07 2.9 2 98.49 97.61 92.09 87.10 0.77 3.48 4.5 3 94.28 100 94.72 96.44 0.72 2.31 3.2 4 98.82 98.18 93.11 92.77 0.24 2.69 11.2 5 98.10 98.53 63.15 84.30 1.78 2.77 1.6 6 98.54 98.33 91.58 76.16 0.69 2.37 3.4 7 98.18 97.78 95.58 94.46 0.88 3.7 4.2 8 99.49 97.93 96.11 92.39 0.83 6.14 7.4 9 99.09 97.64 96.75 96.55 0.39 2.24 5.7 10 97.53 99.37 84.46 98.44 0.52 1.67 3.2 AVG 98.01 98.32 90.58 91.41 0.82 3.14 4.7

Example 2

(17) In a separate set of experiments, in accordance with an embodiment of the present invention, results were obtained as shown in FIG. 1 and Table 2 below. Peripheral blood was extracted from ten human volunteers for use in ten experiments. A CCP was generated in accordance with protocols described herein (see Example 1). Results in FIG. 1 and in Table 2 show the fluorescent intensity of CD31Bright cells in the CCP. CD31 brightness (Dim or Bright) is defined as the ratio between intensity resulting from staining using anti-CD31 FITC-conjugated monoclonal antibodies and intensity resulting from staining using isotype control FITC-conjugated antibodies.

(18) FIG. 1 is a graph showing results obtained from CCP cells in one representative experiment, in accordance with an embodiment of the present invention. CCP cells stained using isotype control FITC-conjugated antibodies are represented by the dashed line and CCP cells stained using FITC-conjugated anti-CD31 antibodies are represented by the black line. Three different intensity areas were marked:

(19) (a) M1low intensity corresponding to non-specific staining of isotype control or cells that do not express CD31, located at geometric mean intensity of 5.38;

(20) (b) M2-dim intensity corresponding to cells expressing CD31 at a geometric mean intensity of 46.69; and

(21) (c) M3-bright intensity corresponding to cells expressing CD31 at a geometric mean intensity of 478.45.

(22) Table 2 is a numerical summary of intensities M1, M2 and M3 and their respective ratios resulting from ten independent experiments.

(23) TABLE-US-00002 TABLE 2 CD31 Intensity Geo Mean Isotype EXP Control dim bright Intensity Ratio No. M1 M2 M3 M2/M1 M3/M1 M3/M2 1 5.25 42.90 299.92 8 57 7 2 5.38 46.69 478.45 9 89 10 3 5.52 30.37 340.24 6 62 11 4 4.9 28.41 266.46 6 54 9 5 4.57 33.19 456.80 7 100 14 6 5.31 34.94 384.76 7 72 11 7 2.91 25.45 318.20 9 109 13 8 2.19 27.43 361.86 13 165 13 9 3.86 33.57 310.46 9 80 9 10 5.3 42.68 400.03 8 75 9 AVG 4.52 34.56 361.72 8.00 86.50 10.69 SE 0.37 2.30 21.74 0.63 10.42 0.66 CD31Bright cells (M3) mean intensity is 86.5 (SE = 10.42) times greater than the negative control intensity (M1) and 10.69 (SE = 0.66) times more than CD31Dim cells (M2) (which themselves have an intensity 8.00 (SE = 0.63) times more than M1). Thus, results indicate that the CCP was enriched to provide CD31+ cells.

Example 3

(24) In a separate set of experiments, in accordance with an embodiment of the present invention, results were obtained as shown in Table 3 below. Peripheral blood was extracted from nine human volunteers for use in nine experiments. A CCP was generated in accordance with protocols described hereinabove with reference to Example 1.

(25) TABLE-US-00003 TABLE 3 % CD31Bright Exp No. S. Cells CCP Enrichment Factor 1 10.1 60.4 6.0 2 25.4 80.85 3.2 3 19.1 76.85 4.0 4 25.1 77.3 3.1 5 16.1 75.8 4.7 6 12.7 75.0 5.9 7 17.5 53.3 3.1 8 21.9 80.96 3.7 9 18.6 64.58 3.5 AVG 18.5 71.67 4.13
Results in Table 3 indicate percentage enrichment of CD31Bright cells in the CCP as compared to the source cells.

Example 4

(26) In a separate set of experiments, a human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP; the CCP was grown on fibronectin or plasma-coated T75 flasks in the presence of medium containing autologous serum (>=10%), 2 ng/ml VEGF, and 5 IU/ml Heparin.

(27) FIG. 2 is a photograph showing the morphology of a typical angiogenic cell precursor (ACP) population, produced in the experiments of Example 3, in accordance with an embodiment of the present invention. Typically, elongated and spindle-shaped cells are observed in cultures of ACPs. This image was obtained from 200 magnification of cultured ACPs.

Example 5

(28) In a separate set of experiments, a human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP, as described hereinabove with respect to Example 4. The CCP was grown on fibronectin or plasma-coated T75 flasks in the presence of medium containing autologous serum (>=10%), 2 ng/ml VEGF, and 5 IU/ml Heparin.

(29) FIG. 3 contains photographs of a typical angiogenic cell precursor (ACP) population, produced in the experiments of Example 2, in accordance with an embodiment of the present invention. Harvested cells were loaded on a glass slide and fixed prior to their specific staining Stained cells were mounted using a fluorescent mounting solution containing the nuclear stain DAPI. FIGS. A1-A3 are a series of photographs from cells stained with FITC-conjugated Ulex-Lectin, cells stained with PE-conjugated anti-CD31, or cells that stained with both Ulex-Lectin and anti-CD31, in accordance with respective embodiments of the present invention. A1 is a photograph of cells stained with the nuclear marker DAPI. A2 is a photograph showing green emission resulting from staining of the same cells with FITC-conjugated Ulex-lectin. A3 is a photograph showing red emission resulting from staining of the same cells with PE-conjugated anti-CD31 antibodies. FIGS. B1-B3 are a series of photographs from cells stained with isotype control antibodies, in accordance with respective embodiments of the present invention. B1 is a photograph of cells stained with the nuclear marker DAPI, B2 is a photograph showing green emission resulting from staining of the same cells with FITC-conjugated mouse IgG antibodies, and B3 is a photograph showing red emission resulting from staining of the same cells with PE-conjugated mouse IgG Antibodies.

(30) Typically, ACP cells fluoresce both red and green indicating adhesion of both Ulex-Lectin and anti-CD31 thereto. Images were obtained from 200 magnification.

Example 6

(31) In a separate set of experiments, a human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP, as described hereinabove with reference to Example 4. The CCP was grown on fibronectin or plasma-coated T75 flasks in the presence of medium containing autologous serum (>=10%), 2 ng/ml VEGF, and 5 IU/ml Heparin.

(32) FIG. 4 contains photographs of a typical angiogenic cell precursor (ACP) population, produced in the experiments of Example 4, in accordance with an embodiment of the present invention. Harvested cells were loaded on a glass slide and fixed prior to their specific staining Stained cells were mounted with a fluorescent mounting solution containing the nuclear stain DAPI. FIGS. A2-A3 are a series of photographs demonstrating uptake of Ac-LDL, cells stained with anti-CD31, or cells that show both uptake of Ac-LDL and staining with anti-CD31, in accordance with respective embodiments of the present invention. A1 is a photograph of cells stained with the nuclear marker DAPI, A2 is a photograph showing green emission resulting from uptake of FITC labeled-Ac-LDL by the same cells, and A3 is a photograph showing red emission resulting from staining of the same cells with PE-conjugated anti-CD31 antibodies. FIGS. B1-B3 are a series of photographs from cells stained with isotype control antibodies, in accordance with an embodiment of the present invention. B1 is a photograph of cells stained with the nuclear marker DAPI, B2 is a photograph showing green emission resulting from staining of the same cells with FITC-conjugated mouse IgG antibodies, and A3 is a photograph showing red emission resulting from staining of the same cells with PE-conjugated mouse IgG Antibodies.

(33) Typically, ACP cells fluoresce both green and red indicating that ACPs uptake Ac-LDL as well as comprise CD31. Images were obtained from 200 magnification.

Example 7

(34) In the same set of experiments, the human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP as described hereinabove with respect to Example 4. Flow-cytometry percentage staining results from nine independent experiments are summarized in Table 4, and show the average staining results obtained on day 5 of culturing.

(35) TABLE-US-00004 TABLE 4 Number Average on Standard experiments (n) day 5 Error % CD34 9 53.1 6.9 % KDR 9 2.3 1.1 % Tie-2 9 6.6 1.6 % Ac-LDL CD31Bright 9 60.7 4.7
Results using such a protocol typically yield a PCP having an average of 60.7% of cells that both demonstrate uptake of Ac-LDL and stain for CD31Bright.

Example 8

(36) In a separate set of experiments, a human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP, as described hereinabove with respect to Example 4. Harvested ACP-rich PCP cells were washed from culture medium and incubated for 24 hours in a serum-free medium. Average secretion levels (pg/ml) of IL-8, VEGF, and angiogenin as obtained from four independent experiments are summarized in Table 5.

(37) TABLE-US-00005 TABLE 5 Group IL-8 pg/ml VEGF pg/ml Angiogenin pg/ml Control Medium 20 20 20 ACP derived medium 10107 165 615

Example 9

(38) In the same set of experiments, a human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP, as described hereinabove with reference to Example 4. Angiogenic pattern and vascular tube formation of ACP-rich PCP cells were examined microscopically following plating of the cells on an extracellular matrix gel (ECM). Typically, semi-closed and closed polygons of capillaries and complex mesh-like capillary structures were observed and scored according to a scale published by Kayisli et al. (52) as grade 4-5, indicating the angiogenic-inducing properties of the ACP-rich PCP.

(39) FIG. 5 is a photograph showing tube formation in an ACPs, produced in the experiments of Example 6, in accordance with an embodiment of the present invention. Typical mesh-like capillary structures generated from a harvested ACPs are present in the culture and are suitable for administration to a human.

Example 10

(40) In a separate set of experiments, a human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP; the CCP was grown on fibronectin or plasma-coated T75 flasks in the presence of medium containing autologous serum (>=10%), 2-10 ng/ml VEGF, and 5 IU/ml Heparin. At the end of the culturing period, ACP cells were harvested and labeled with 0.8 ug/ml Ac-LDL-DiO for 15 min at 37 C and placed in inserts which were placed in wells. One million labeled ACPs were placed on microporous membrane inserts with a pore size of 8 micrometer. 200 ul medium was placed at the bottom of each of the wells. Negative control (M199), positive control (e.g., 20 ng/ml VEGF, 20 ng/ml bFGF, and 20 ng/ml SCF) and 0.08-60 ng/ml human recombinant Interleukin-8 (hIL-8) diluted in M199 medium were plated in respective wells and the ACP cells were allowed to migrate toward each respective medium. Following 1 hour incubation in the presence of the negative control medium, the positive control medium, and the IL-8 containing media, labeled migrating cells from 10-15 random microscopic fields were evaluated using fluorescent microscope and automated counting software (NIH ImageJ). Calculation of cell number per 1 mm^2 was based on area of counting field (20) which equals 0.178 mm^2, and each mm^2 contains 5.62 fields. Assessment of ACP migratory potential indicated that ACPs migrate toward chemokines such as VEGF, bFGF, SCF, and hIL-8 in a manner dependent on respective concentrations thereof, e.g., hIL-8 concentration of typically higher than 6.7 ng/ml induces substantial migration of ACPs, and hIL-8 concentrations of about 7-20 ng/ml typically induce substantial migration of ACPs.

(41) FIGS. 6A and 6B are graphs showing results obtained in five experiments of Example 10, in accordance with an embodiment of the present invention. FIG. 6A shows migration toward negative and positive control media of Ac-LDL-DiO pre-labeled ACPs. FIG. 6B shows a dosage-dependence curve reflecting migration of Ac-LDL-DiO pre-labeled ACPs in response to increasing concentrations of hIL-8 ACPs derived from the human-PBMC-derived CCP show statistically significant migration toward the positive control samples. Moreover, ACP migration corresponding to increasing hIL-8 doses was observed. Dose-dependent ACP migration peaked at 6.7-20 ng/ml of hIL-8.

Example 11

(42) In a separate set of experiments, the human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP, as described hereinabove with reference to Examples 4 or 10. In some embodiments, generation of the ACP-rich PCP is attributed to migration of ACP cells to a specific chemokine, in combination with the differentiation of CCP cells. Migratory potential of ACP-rich PCP was measured as described hereinabove with respect to Example 10. In this example, a conditioned medium (CM) was generated using the patients' cells which secrete chemokines into the medium. The patients' cells were then extracted from the medium, leaving a chemokine-rich medium for subsequent plating of ACP therein. The potential for ACP migration in response to chemokines was then assessed when the ACPs were incubated for 1 hour with conditioned medium.

(43) Following 1 hour incubation in the presence of negative control (M199); 20 ng/ml hIL-8; or CM (at concentrations of 2-20 ng/ml), migration of labeled cells from 10-15 random microscopic fields was evaluated using a fluorescent microscope and automated counting software (NIH ImageJ). Calculation of cell number per 1 mm^2 is based on area of counting field (20)=0.148 mm^2 and thus each square millimeter contains 6.7 fields. It was determined that ACPs migrate toward chemokines secreted during the production of the ACP-rich PCP.

(44) For some applications, the generated ACP-rich PCP batches were used to treat cardiovascular patients. All patients treated with these batches showed more than 10% improvement in left-ventricular-ejection fraction both 3 and 6 months following treatment. It is hypothesized that this improvement was enabled at least in part by the migration of ACPs to the vicinity.

(45) FIG. 7 is a graph showing results obtained in four experiments of Example 11, in accordance with an embodiment of the present invention. The ACPs derived from the human-PBMC-derived CCP show statistically significant migration toward the conditioned medium containing secreted chemokines; this medium was generated in the process of the production of ACP-rich PCP.

Example 12

(46) In a separate set of experiments, migratory potential of human-PBMC toward hIL-8 was measured. In vitro assessment of PBMC migratory capability in response to hIL-8 was used to determine the potential of IL-8 to mobilize blood derived stem/progenitor cells from peripheral blood to locations in which high concentrations of IL-8 are expressed in vivo. Peripheral blood was extracted from six human volunteers for use in six respective experiments. In each experiment, a Ficoll gradient was used to generate a population of PBMCs. One million PBMCs were placed on 3 um pore size microporous membrane inserts which were placed in wells. 200 ul medium was placed at the bottom of each of the wells. Negative control (M199) and positive control (20 ng/ml hIL-8) diluted in M199 medium were plated in respective wells and the PBMCs cells were allowed to migrate toward each respective medium. Following 1 hour incubation in the presence of the negative control medium and the positive control medium, migration of cells from 10-15 random microscopic fields was evaluated using a fluorescent microscope and automated counting software (NIH ImageJ). Calculation of cell number per 1 mm^2 is based on area of counting field (20) which equals 0.148 mm^2, and each square millimeter contains 6.7 fields. It was determined that hIL-8 induced mobilization of only a small fraction of the PBMCs, probably the stem/progenitor cells.

(47) FIG. 8 is a graph of migration of PBMCs in response to hIL-8, in accordance with an embodiment of the present invention. The results were obtained from six experiments (Example 12), and show that stem/progenitor cells derived from human-PBMCs migrate toward hIL-8.

Example 13

(48) Reference is now made to FIGS. 9A and 9B which are graphs showing results obtained in the experiments following injection into rats of ACP-rich PCPs derived from a human-PBMC-derived CCP (as described hereinabove with respect to Example 4) following acute myocardial infarction, in accordance with an embodiment of the present invention.

(49) The human-PBMC-derived CCP was cultured in order to generate an ACP-rich PCP as described in Example 4. ACP-rich PCP therapeutic potential was then assessed in a rat model of acute myocardial infarction which was induced in 15 male nude rats (200-225 g) by ligation of the left anterior descending (LAD) artery. Six days after myocardial infarction, 10 rats were injected with 1.510^6 ACP-enriched cells (ACP, n=10), while 5 rats were injected with the culture medium (Control, n=5), via the aortic arch. Cardiac function (ejection fraction) and the ratio of necrotic scar area to left ventricular free wall area were measured 28 days following the ACP-rich PCP and the culture medium administrations. It is to be noted that the percentage of ejection fraction of the ACP-administered rats, as represented by FIG. 9A, increased substantially in comparison to the decreased percentage ejection fraction of the control rats. Additionally, a percentage reduction of necrotic tissue was observed in the ACP-administered rats in comparison to the percentage of necrotic tissue observed in the control rats. Paraffin fixed tissue sections obtained from the 10 ACP-administered rats were stained in order to trace engrafted human cells and cardiomyocyte (CMC) markers in the border area of the scar tissue.

(50) FIGS. 9C, 9D and 9E are photographs showing typical sections taken from a heart of one of the 10 rats 28 days after the injection of the ACPs derived from a human-PBMC-derived CCP in the experiments of the present example (Example 13) in accordance with an embodiment of the present invention. FIG. 9C shows staining of the rat's heart cells with anti-human mitochondria. FIG. 9D shows the cells stained for CMC markers (myosin heavy chain (MHC)), FIG. 9E shows the rat heart cells stained for cardiac Troponin I. (Reference is again made to FIG. 9C-E. The stained cells are marked by arrows). These results depicted in FIGS. 9C-D demonstrate that the human ACPs, derived in accordance with an embodiment of the present invention from the hPBMC-derived CCP, homed to damaged cardiac tissues, engrafted, and is hypothesized to have transdifferentiated into cells expressing cardiomyocyte markers.

(51) It is to be noted that ACPs typically improve systemic endothelial functioning, as expressed by improved ejection fraction and reduced necrosis. Particular examples of improvement due to administration of ACPs, derived in accordance with an embodiment of the present invention, include improved cardiovascular functioning and improved sexual functioning. The scope of the present invention includes identifying a patient having cardiovascular dysfunction or sexual dysfunction, and administering ACPs to the patient in order to treat the dysfunction.

Example 14

(52) In a production procedure, individual autologous human-PBMC-derived CCPs were cultured in order to generate an ACP-rich PCP, as described hereinabove. The CCPs were grown on autologous plasma-coated T75 flasks in the presence of medium containing autologous serum (>=10%), 2-10 ng/ml VEGF, and 5 IU/ml Heparin. Harvested cells, approved by Quality Control for clinical use, were administrated to patients. The therapeutic potential of ACP-rich PCP is summarized in results of administration thereof to 14 patients suffering from end-stage heart failure. Left ventricular ejection fraction (EF) and disease severity score (Score) were assessed prior to and 1-8 months after the ACP cell administration. Improvement of these parameters was calculated relative to each patient's baseline evaluation according to the following equation:
% Improvement=(Test result after treatmentBaseline test result)/Baseline test result.

(53) Results show statistically significant improvement (p<0.0001; tested using two-tailed, paired t test analysis) in both parameters following treatment by administering ACP-rich PCP.

(54) Table 6 shows the number of treated patients, averages and individual results relating to EF and disease severity score, as well as the calculated percent improvement thereof.

(55) TABLE-US-00006 TABLE 6 % EF* SCORE* % EF At 1-8 EF % SCORE At 1-8 SCORE % Batch No. Baseline Months Improvement Baseline Months Improvement N 14 14 14 14.0 14.0 14.0 Average 24.1 34.8 49.8 2.9 1.6 45.6 Range 14.9-36.0 20.0-50.0 11.1-133 2.0-4.0 1.0-3.0 29.0-67.0 SE 2.1 2.8 10.9 0.1 0.1 4.1 PCEPC066 30.0 40.0 33.3 2.00 1.00 50.00 PCEPC081 23.0 50.0 117.4 3.00 1.00 66.67 PCEPC083 27.5 32.5 18.2 3.00 2.00 33.33 PCEPC091 14.9 20.0 34.2 3.00 2.00 33.33 PCEPC092 35.0 41.0 17.1 3.00 2.00 33.33 PCEPC094 36.0 40.0 11.1 3.00 2.00 33.33 PCEPC097 15.0 27.5 83.3 3.00 1.00 66.67 PCEPC099 15.0 35.0 133.3 3.50 2.00 42.86 PCEPC103 18.3 20.9 14.2 3.00 2.00 33.33 PCEPC106 15.0 20.0 33.3 3.00 1.00 66.67 PCEPC110 25.0 50.0 100.0 3.00 2.00 33.33 PCEPC114 22.0 30.0 36.4 2.00 1.00 50.00 PCEPC121 25.0 32.0 28.0 3.50 2.50 28.57 PCEPC137 35.0 48.0 37.1 3.00 1.00 66.67 *Significant improvement p < 0.0001

Example 15

(56) In a separate set of experiments, a human-PBMC-derived CCP was cultured in order to generate a cardiomyocyte (CMC)-rich PCP; the CCP was grown on fibronectin or plasma-coated T75 flasks in accordance with protocols described herein (see medium preparation).

(57) FIG. 10 is a photograph of a typical CMC-rich PCP from the experiments of the current example (Example 15), derived in accordance with an embodiment of the present invention. Typically, these cells appear elongated with dark cytoplasm, which may indicate high protein content. This image was obtained from 200 magnification of cultured CMC-rich PCP cells.

(58) FIGS. 11A, 11B, and 11C are photographs showing immunostaining of CCP-derived cardiomyocytes in the experiments of the current example (Example 15), in accordance with an embodiment of the present invention. Slide-fixed CMC PCP cells were stained with:

(59) FIG. 11Aanti-cardiac Troponin detected by anti-mouse Cy-3;

(60) FIG. 11Banti-alpha-actin detected by anti-mouse IgG-FITC; and

(61) FIG. 11Canti-connexin 43 detected by anti-mouse IgG-FITC.

(62) Cells stained with non-specific mouse IgG were detected by anti-mouse IgG-FITC or by anti-mouse IgG-Cy3 and were used as negative controls.

(63) FIGS. 11A-C show that CMC-rich PCP cells expressed the typical cardiomyocyte cellular markers: cardiac Troponin T (FIG. 11A), alpha-actin (FIG. 11B), as well as the functionally important GAP junction marker connexin-43 (FIG. 11C). The images were obtained from 100 magnification of slide-fixed cells.

Example 16

(64) In the same set of experiments that produced the results shown in FIGS. 10-11C, a human-PBMC-derived CCP was cultured in order to generate a CMC-rich PCP; the CCP was grown on fibronectin or plasma-coated T75 flasks in accordance with protocols described herein (see medium preparation).

(65) FIGS. 12A and 12B are graphs showing flow cytometry analysis results obtained from immunostaining of a CMC-rich PCP in the experiments of the current example (Example 16), in accordance with respective embodiments of the present invention. In FIGS. 12A-B, lines describing control, e.g., non-specific staining, are marked as Control; specific immunostaining with the cardiac cellular markers desmin and troponin T are marked as Desmin (FIG. 12A) and Troponin T (FIG. 12B), respectively. The M1 line represents the statistical marker area in which the cells are positively stained for the respective marker.

Example 17

(66) In a separate set of experiments, a human-PBMC-derived CCP was cultured in order to generate a CMC-rich PCP, as described hereinabove. The CMC-rich PCP cells' therapeutic potential was assessed in a rat model of acute myocardial infarction. CMC-rich PCP cells were used for implantation into a rat model of induced acute myocardial infarction as described hereinabove with respect to Example 13 (with the exception that CMC-rich PCP cells were used for implantation into the rat model in the current example, whereas in Example 13, ACP-rich PCP cells were used for implantation). Six days after myocardial infarction, heart muscle of 9 rats were injected with 1.510^6 CMC PCP cells (CMC, n=9), while heart muscle of 5 rats were injected with culture medium (Control, n=5). Cardiac function (ejection fraction) was evaluated 14 days following the administration of the CMC-rich PCP cells or culture medium.

(67) FIG. 13 is a graph showing experimental results obtained in the experiments of Example 13, in accordance with another embodiment of the present invention. It is to be noted that the percentage of ejection fraction of the CMC-administered rats increased substantially in comparison to the decreased percentage ejection fraction of the control rats.

(68) A series of protocols are described hereinbelow which may be used, as appropriate, separately or in combination with Examples 1-17, in accordance with embodiments of the present invention. It is to be appreciated that numerical values are provided by way of illustration and not limitation. Typically, but not necessarily, protocols may be derived using values selected from a range of values that is within 20% of the value shown. Similarly, although certain steps are described herein with a high level of specificity, a person of ordinary skill in the art will appreciate that additional or other steps may be performed, mutatis mutandis.

(69) In accordance with an embodiment of the present invention, generation of a single-cell suspension is carried out using the following protocols:

(70) PROTOCOL 1: Extraction of peripheral blood mononuclear cells (PBMC). Receive blood bag and sterilize it with 70% alcohol. Load blood cells onto a Ficoll gradient. Spin the tubes for 20 minutes at 1050 g at room temperature (RT), with no brake. Collect most of the plasma from the supernatant. Collect the white blood cell fraction from every tube. Transfer the collected cells to a new 50 ml tube, adjust volume to 30 ml per tube using PBS. Spin tubes for 15 minutes at 580 g, RT, and discard supernatant. Count cells in Trypan Blue. Re-suspend in culture medium comprising, for example, X-vivo 15.

(71) PROTOCOL 2: Extraction of cells from umbilical cord. Isolate 10 cm umbilical cord. Wash thoroughly with sterile PBS. Identify the major vein of the cord, and clamp one end of the vein. Wash twice with 30 ml sterile PBS. Fill vein with 0.15% collagenase (about 5 ml of 0.15% collagenase solution). Clamp the second end of the vein. Incubate at 37 C for 15 min. Wash outer side of the cord with 70% ethanol. Release the clamp from one end of the vein and collect the cell suspension. Centrifuge for 10 min at 580 g, 21 C. Re-suspend the cells in culture medium comprising, for example, X-vivo 15, 10% autologous serum, 5 IU/ml heparin, and one or more growth factors.

(72) PROTOCOL 3: Extraction of cells from bone marrow. Get bone marrow aspiration from surgical room. Re-suspend in culture medium comprising, for example, X-vivo 15, 10% autologous serum, 5 IU/ml heparin, and one or more growth factors. Pass suspension through a 200 um mesh.

(73) In accordance with an embodiment of the present invention, generation of a CCP is carried out using the following protocols:

(74) PROTOCOL 1: Generation of a human CCP from PBMCs using a Percoll gradient. Prepare gradient by mixing a ratio of 5.55 Percoll (1.13 g/ml): 3.6 ddH2O:1 PBS10. For every 50 ml tube of Percoll: mix 20 ml of Percoll stock, 13 ml of ddH2O and 3.6 ml of PBS10. Mix vigorously, by vortexing, for at least 1 min. Load 34 ml mix into each 50 ml tube. Centrifuge tubes, in a fixed angle rotor, for 30 min at 17,000 g, 21 C, with no brake. Gently layer 3.0 ml of cell suspension of 150 million-400 million PBMCs on top of the gradient. Prepare a second tube with density marker beads: gently layer 3.0 ml of medium on top of the gradient. Gently load density marker beads10 ul from each bead type. Centrifuge tubes, in a swinging bucket rotor, for 30 min at 1260 g at 13 C, with no brake. Gently collect all bands located above the red beads, and transfer to tube with 10 ml medium. Centrifuge cells for 15 min at 580 g at 21 C. Discard supernatant and re-suspend pellet in medium. Count cells in Trypan blue. Centrifuge cells for 10 min at 390 g, 21 C. Discard supernatant and re-suspend pellet in medium. Take CCP cells for FACS staining

(75) PROTOCOL 2: Generation of human CCP from PBMCs using an OptiPrep gradient. Take up to 130 million cells for each enrichment tube. Spin cells for 10 min at 394 g, 21 C. Suspend cell pellet in 10 ml of donor serum. Prepare a 1.068 g/ml OptiPrep gradient by mixing a ratio of 1 OptiPrep:4.1 PBS. For every 50 ml enrichment tube: Mix 10 ml of cell suspension with 4 ml OptiPrep. For preparation of a 1.068 g/ml OptiPrep gradient, mix 5 ml of OptiPrep and 20.5 ml of PBS. Gently layer 20 ml of the 1.068 g/ml gradient on top of the cell suspension. Gently layer 1.5 ml Hank's buffered saline (HBS) on top of the gradient layer. Centrifuge for 30 min at 700 g at 4 C, with no brake. Gently collect the layer of cells that floats to the top of the 1.068 g/ml OptiPrep gradient into a 50 ml tube pre-filled with PBS. Centrifuge for 10 min at 394 g, 21 C. Discard supernatant and re-suspend pellet in medium. Count cells in Trypan Blue.

(76) It is to be noted that culture containers are typically either un-coated or coated with one or a combination of ACP-enhancing materials such as collagen, fibronectin, CD34, CD133, Tie-2, or anti-CD117.

(77) In accordance with an embodiment of the present invention, the coating of a tissue culture container is carried out using the following protocols:

(78) PROTOCOL 1: Coating T75 flasks with 25 ug/ml fibronectin. For 20 T75 flasksPrepare up to seven days before, or on day of PBMC preparation. Prepare 50 ml of 25 ug/ml fibronectin solution in PBS. Fill every flask with 2-5 ml fibronectin 25 ug/ml. Incubate at 37 C for at least 30 min. Collect fibronectin solution. Wash flask twice in PBS. Dry flasks Keep dry flasks at room temperature. Dried flasks can be saved for one week at room temperature (RT).

(79) PROTOCOL 2: Coating T75 flasks with 25 ug/ml fibronectin and 5 ng/ml BDNF Coat flasks with Fibronectin 25 g/ml, as described in Protocol 1. Prepare 50 ml of 5 ng/ml BDNF solution in PBS. After washing off Fibronectin, fill every flask with 2-5 ml BDNF 10 ng/ml. Incubate at 37 C for 1 hour. Collect the solution. Wash flask twice in 10 ml PBS. Keep dry flasks at room temperature until use.

(80) In accordance with an embodiment of the present invention, serum preparation is carried out using the following protocol: (Serum can be obtained directly or prepared from plasma).

(81) PROTOCOL: Preparation of serum from human plasma. Take 100 ml of undiluted blood. Spin at 1100 g (2500 rpm) for 10 min. Transfer the upper layer (plasma) to a new 50 ml tube. Add 1.0 ml 0.8M CaCl.sub.2-2H.sub.2O for every 40 ml plasma. Incubate for 0.5-3 hours at 37 C. Spin coagulated plasma 5 min at 2500 g. Collect the serum in a new tube, avoiding clotting. Aliquot collected serum and save at 20 C until use.

(82) In accordance with an embodiment of the present invention, medium preparation is carried out using the following protocols:

(83) Medium should contain 1-20% autologous serum and/or 1-20% conditioned medium.

(84) Medium can contain one or more additives, such as LIF, EPO, IGF, b-FGF, M-CSF, GM-CSF, TGF alpha, TGF beta, VEGF, BHA, BDNF, NGF, EGF, NT3, NT4/5, GDNF, S-100, CNTF, NGF3, CFN, ADMIF, estrogen, progesterone, cortisone, cortisol, dexamethasone, or any other molecule from the steroid family, prolactin, an adrenocorticoid hormone, ACTH, glutamate, serotonin, acetylcholine, NO, retinoic acid (RA) or any other vitamin D derivative, Heparin, insulin, forskolin, Simvastatin, MCDB-201, MCT-165, glatiramer acetate, a glatiramer acetate-like molecule, IFN alpha, IFN beta or any other immunoregulatory agent sodium selenite, linoleic acid, ascorbic acid, transferrin, 5-azacytidine, PDGF, VEGF, cardiotrophin, and thrombin or Rosiglitazone in various concentrations, typically ranging from about 100 pg/ml to about 100 g/ml (or molar equivalents).

(85) Typically, medium should not be used more than 10 days from its preparation date.

(86) PROTOCOL 1: Medium for enhancement of CCP-derived angiogenic cell precursors (ACPs). Serum-free medium (e.g., X-vivo 15) 10% autologous serum 5 IU/ml Heparin 5 ng/ml VEGF 1 ng/ml EPO

(87) PROTOCOL 2: Medium for enhancement of CCP-derived neuronal progenitor cells. Serum-free medium (e.g., X-vivo 15) 20 ng/ml bFGF 50 ng/ml NGF 200 uM BHA (this is added during the last 24 hours of culturing) 10 ng/ml IFN beta 10 ug/ml glatiramer acetate 10 uM forskolin 1 uM cortisone 1 ug/ml insulin

(88) PROTOCOL 2.1: Medium for enhancement of CCP-derived neuronal progenitor cells. Serum-free medium (e.g., X-vivo 15) 20 ng/ml bFGF 50 ng/ml NGF 25 ng/ml BDNF 200 uM BHA (this is added during the last 24 hours of culturing)

(89) PROTOCOL 3: Medium for enhancement of CCP-derived retinal cells. Serum-free medium (e.g., X-vivo 15)) 10% autologous serum 5 IU/ml Heparin 10 ng/ml EGF 20 ng/ml bFGF 10 ug/ml glatiramer acetate 50 ng/ml NGF3

(90) PROTOCOL 4a. Medium for enhancement of CCP-derived cardiomyocyte (CMC) progenitor cells. Step I Serum-free medium (e.g., X-vivo 15 10% autologous serum 20 ng/ml bFGF 20 ug/ml IFN beta 5 IU heparin. Step II Five to ten days after culture onset, add 3 uM 5-azacytidine for 24 hours.

(91) PROTOCOL 4b: Medium for enhancement of CCP-derived CMC progenitor cells. Serum free medium DMEM-Low glucose 20% autologous serum 10% MCDB-201 2 ug/ml Insulin 2 ug/ml Transferin 10 ng/ml Sodium Selenite 50 mg/ml BSA 1 nM Dexamethasone 20 ug/ml Glatiramer acetate 0.47 ug/ml Linoleic acid 0.1 mM Ascorbic Acid 100 U/ml penicillin

(92) In accordance with an embodiment of the present invention, conditioned medium preparation is carried out using the following protocol:

(93) PROTOCOL 1: Preparation of 100 ml enriched medium containing 10% autologous conditioned medium.

(94) Thaw 10 ml conditioned medium in an incubator.

(95) When thawed, add it to culture medium using pipette. Extraction of tissue pieces for co-culture: Dissection of rat blood vessels (other non-human or human tissues may also be used): Anesthetize animal using anesthetic reagents (e.g., 60-70% CO2, isoflurane, benzocaine, etc.). Lay animal on its back and fix it to an operating table. Using sterile scissors, cut animal's skin and expose the inner dermis. Using a second set of sterile scissors, cut the dermis, cut chest bones, and expose the heart and aorta. Cut small pieces, 0.2-1 cm long, from the aorta and other blood vessels, and place them in a container pre-filled with 50 ml cold culture medium (e.g. RPMI, X-vivo 15, or any other growth medium). Using forceps and scissors, clean tissue sections, to remove outer layers such as muscle, fat, and connective tissue. Using forceps and scalpel, cut each blood vessel along its length, and expose the inner layer of endothelial cells. Using forceps and scalpel, cut small pieces of up to 0.1 cm2 from the tissue.

(96) It is to be understood that whereas this technique is in accordance with one embodiment of the present invention, the scope of the present invention includes extracting a blood vessel from a human, as well. For example, an incision may be made over the saphenous vein, in order to facilitate dissection of a distal 1 cm portion of the vein. Tributary veins thereto are tied and transected. Distal and proximal ends of the 1 cm portion of the saphenous vein are tied, and the vein is harvested.

(97) Use the dissected tissue for direct and/or indirect co-culturing with the CCP and/or to generate conditioned medium.

(98) Generation of conditioned medium: Lay dissected pieces in culture containers, for example in T75 flasks, or 50 ml tubes. Optionally, fill with cell culture medium containing 0.1-3 ug/ml or 3-100 ug/ml apoptotic reagent (such as valinomycin, etoposide or Staurosporine), until all pieces are covered. Refresh culture medium every 2 days. Collect this medium (now conditioned medium) into 50 ml tubes. Spin collected conditioned medium at 450 g for 10 min, at room temperature. Collect supernatant in a new sterile container.

(99) Details regarding preservation of the conditioned medium, in accordance with an embodiment of the present invention, are described hereinbelow.

(100) In accordance with an embodiment of the present invention, culturing of a CCP to produce a PCP is carried out using the following protocols:

(101) PROTOCOL 1: Culturing of CCP cell suspension in T75 Flasks. Spin suspension for 15 minutes at 450 g, 21 C. Discard the supernatant. Gently, mix cell pellet and re-suspend the CCP cells. Re-suspend pellet to 10 million CCP cells/ml. Fill T75 flask with 15 ml enriched medium, and add 5 ml of 10 million CCP cells/ml to attain a final concentration of 50 million CCP cells/flask. Incubate T75 flasks, plates and slides at 37 C, 5% CO2.

(102) PROTOCOL 2: Applied hypoxia.

(103) For some applications, increased expansion and/or differentiation of the CCP may be obtained by exposure of the cell culture to oxygen starvation, e.g., 0.1-5% or 5-15% oxygen (hypoxia), for 2-12 or 12-48 hours. This is typically done one or more times, at different points during cell culturing.

(104) Incubate T75 flasks in an oxygen-controlled incubator. Set the oxygen pressure at 0.1%, and maintain it at this level for 24 hours. Remove the flasks from the incubator and examine the culture. Take a sample of CCP cells and test viability by Trypan blue exclusion method. Set the oxygen pressure of the incubator at 20%. Re-insert the flasks into the incubator and continue incubation for the rest of the period. This procedure can be repeated, for example, once a week during the culture period and/or within 24, 48, or 72 hours before termination of the culture.

(105) PROTOCOL 3: Reseeding of adherent and/or detached and/or floating cells.

(106) For some applications, increased expansion and differentiation of the CCP may be achieved by re-seeding collected cells on new pre-coated dishes in culture medium. Collect all cultured CCP in tubes. Spin tubes for 10 minutes at 450 g, 21 C. Discard the supernatant. Gently mix pellet and re-suspend cells in 10 ml fresh medium per T75 flask. Seed suspended cells in new pre-coated T75 flasks. Continue culturing the cells, and perform all other activities (e.g., medium refreshment, visual inspection, and/or flow cytometry), as appropriate, as described herein.

(107) This procedure can be performed weekly during the culture period and/or within 24, 48, or 72 hours before termination of the culture.

(108) In accordance with an embodiment of the present invention, co-culturing of CCP with tissue-derived conditioned medium is carried out using the following protocol:

(109) PROTOCOL 1: Culturing of CCP in the presence of conditioned medium derived from a blood vessel culture. Spin CCP cells for 15 minutes at 500 g, 21 C. Discard the supernatant. Gently mix cell pellet and re-suspend cells to 5-50 million/ml in autologous medium containing 1-20% autologous serum and/or 1-20% conditioned medium. Seed flasks with 2-5 million CCP cells/ml. Incubate flasks at 37 C, 5% CO2. After first three days of culture, non-adherent cells can be removed from the culture.

(110) In accordance with an embodiment of the present invention, refreshing of the media in ongoing growing CCP cultures is carried out using the following protocol:

(111) Refreshing of the media in ongoing growing flasks should occur every 3-4 days.

(112) PROTOCOL 1: Refreshing of medium in T-75 Flasks. Collect non-adherent cells in 50 ml tubes. Fill every flask with 10 ml fresh culture medium enriched with conditioned medium. Spin tubes for 10 minutes at 450 g, RT; discard the supernatant. Gently mix cell pellet and re-suspend cells in 10 ml/flask fresh culture medium enriched with condition medium. Return 5 ml of cell suspension to every flask.

(113) In accordance with an embodiment of the present invention, indirect co-culture of CCP cells with tissue dissection is carried out using the following protocol:

(114) PROTOCOL 1: Indirect co-culture of dissected blood vessel and CCP cells in a semi-permeable membrane apparatus. Lay dissected tissue pieces in the upper chamber of the apparatus on top of the semi-permeable membrane. Implant CCP cells in lower chamber. Lower chamber can be pre-coated with growth-enhancing molecules such as collagen, plasma, fibronectin, a growth factor, tissue-derived extra cellular matrix and an antibody. Refresh culture medium in the upper chamberaspirate conditioned medium into 50 ml tubes and add autologous culture medium. Preserve collected conditioned medium at 20 C. Remove upper chamber after four days of co-culture. Refresh culture medium of the CCP cells with culture medium containing 1-20% autologous serum and/or 1-20% conditioned medium. Continue growing and harvesting as described herein. Co-culture in separate chambers within a culture container

(115) In accordance with an embodiment of the present invention, co-culturing within a culture container is carried out using the following protocol:

(116) PROTOCOL 1: Direct co-culturing of autologous dissected blood vessel and CCP cells. Lay dissected tissue pieces in pre-coated flasks. Implant CCP cells in pre coated second chamber. Using forceps, take out tissue pieces after four days of co-culture. Refresh culture medium of the CCP cells with culture medium containing 1-20% autologous serum and/or 1-20% condition medium. Continue growing and harvesting as described herein.

(117) In accordance with an embodiment of the present invention, harvesting of the cellular product is carried out using the following protocol:

(118) PROTOCOL 1: Collection of resulting ACP cultures. Collect cells in 50 ml tubes. Carefully wash flask surface by pipetting with cold PBS to detach adherent cells. Collect washed adherent cells to 50 ml tubes. Add 5 ml of cold PBS. Detach remaining adherent cells using gentle movements with cell scraper. Collect the detached cells and add them to the tubes Optionally, add 5 ml EDTA to each flask and incubate at 37 C for 5 min. Collect the detached cells and add them to the tubes Spin tubes for 5 min, at 450 g, room temperature. Re-suspend the pellets in 2-5 ml PBS. Count the cells in Trypan blue.

(119) In accordance with an embodiment of the present invention, cellular product preservation is carried out using the following protocols:

(120) Cellular product can be kept in preservation media or frozen in freezing buffer until use for transplantation into a patient.

(121) PROTOCOL 1: Cryopreservation of cellular product.

(122) Prepare freezing buffer containing 90% human autologous serum and 10% DMSO. Suspend cellular product in freezing buffer and freeze in liquid nitrogen.

(123) PROTOCOL 2: Short-period preservation of cellular product.

(124) Prepare preservation medium including growth medium containing 1-20% autologous serum, with few or no other additives. Maintain preservation medium with cellular product at 2-12 C

(125) In accordance with an embodiment of the present invention, conditioned medium collection and preservation is carried out using the following protocol: Conditioned medium can be kept until use for growth medium preparation. Conditioned medium should be collected under sterile conditions. Spin collected conditioned medium for 10 min at 450 g, 21 C. Collect supernatant in a new sterile container. Filter supernatant through a 22 um membrane. Aliquot conditioned medium to 10 and/or 50 ml sterile tubes, pre-marked with donor details. Keep at 20 C until use.

(126) In accordance with an embodiment of the present invention, FACS staining is carried out using the following protocol:

(127) PROTOCOL 1: Staining of ACP enriched population.

(128) FACS staining protocol:

(129) TABLE-US-00007 Tube No. Staining Aim of staining 1. Cells Un-stained control 2. CD45 (IgG1)-FITC Single staining for PMT and 3. CD14-PE (IgG2a) compensation settings 4. CD45 (IgG1)-APC 5. mIgG1-FITC Isotype control mIgG1-PE mIgG1-APC 6. CD45-FITC (IgG1) KDR-PE (IgG2a) CD34-APC (IgG1) 7. Ac-LDL-FITC CD31-PE (IgG1) 8. Ulex-Lectin-FITC CD31-PE (IgG1) 9. mIgG1-FITC Isotype control mIgG2a-PE mIgG1-APC 10. CD45-FITC (IgG1) CD133-PE (IgG2a) CD34-APC (IgG1)

(130) PROTOCOL 2: Staining of CMC progenitors.

(131) FACS staining protocol for fixed permeabilized cells:

(132) TABLE-US-00008 Staining Staining Tube No. 1.sup.st step 2.sup.nd step Aim of staining 1 Cells Un-stained control 2 CD45-FITC Single staining for (IgG1) PMT and compensation 3 CD14-PE settings (IgG2a) 5 mIgG1 Anti mouse -PE Isotype control 6 Desmin Anti mouse -PE 7 Troponin T Anti mouse -PE Isotype control

(133) In accordance with an embodiment of the present invention, immunohistochemistry staining (IHC) is carried out using the following protocols:

(134) PROTOCOL 1: IHC staining protocol for ACPs.

(135) TABLE-US-00009 Staining Slide No. 1st step Aim of staining 1. mIgG1 Isotype control 2. mIgG1-PE Isotype control 3. CD34-APC Specific Staining 4. CD144-FITC Specific Staining 5. CD133-PE Specific Staining 6. Ac-LDL-FITC Specific Staining CD31-PE 7. Ulex-Lectin-FITC Specific Staining CD31-PE

(136) PROTOCOL 2: IHC staining protocol for CMC progenitors.

(137) TABLE-US-00010 Staining Staining Slide No. 1st step 2nd step Aim of staining 1. mIgG1-FITC Isotype control 2. mIgG1 Anti mouse-Cy-3 Isotype control 3. Connexin 43 Anti mouse-FITC Specific Staining 4. Alfa actin Anti mouse-FITC Specific Staining 5. Troponin Anti mouse-PE Specific Staining

(138) In accordance with an embodiment of the present invention, a tube formation assay is carried out using the following protocol:

(139) Tube formation was tested using the ECM625(Chemicon) in vitro angiogenesis assay kit.

(140) Angiogenic pattern and vascular tube formation was numerically scored as described by Kayisli U. A. et al. 2005 (52).

(141) In accordance with an embodiment of the present invention, secretion of cytokines from harvested cells is assessed using the following protocols: Culture 0.5-110^6 cells/ml over night in 24 well plates in serum-free medium (e.g., X-vivo 15) Collect culture supernatant and spin at 1400 rpm for 5 minutes Transfer supernatant to an eppendorf tube and freeze at 80 C until ready to test cytokine secretion.

(142) PROTOCOL 1: ELISA for IL-8. A commercial DuoSet CXCr8/IL-8 (R&D Systems) was used for the detection of IL-8 secretion.

(143) PROTOCOL 2: Cytometric Bead Array. A commercial cytometric bead array (CBA) kit for human angiogenesis (BD 558014) was used for the detection of IL-8, VEGF, TNF and Angiogenin secretion.

(144) It is to be noted that the scope of the present invention includes injecting IL-8 into a human patient in order to recruit ACP cells to a given destination within a given patient, in accordance with the needs of the patient.

(145) For some applications, techniques described herein are practiced in combination with techniques described in one or more of the references cited in the Background section and Cross-References section of the present patent application. All references cited herein, including patents, patent applications, and articles, are incorporated herein by reference.

(146) It is to be appreciated that by way of illustration and not limitation, techniques are described herein with respect to cells derived from an animal source. The scope of the present invention includes performing the techniques described herein using a CCP derived from non-animal cells (e.g., plant cells), mutatis mutandis.

(147) It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove. Rather, the scope of the present invention includes both combinations and subcombinations of the various features described hereinabove, as well as variations and modifications thereof that are not in the prior art, which would occur to persons skilled in the art upon reading the foregoing description.